Exoglycosidases are enzymes which can modify cell membrane carbohydrate epitopes and thereby modify the immune response. More particularly, such enzymes can be used in the process of converting types A, B, and AB, red blood cells to universally transfusible type O cells. The U.S. Pat. No. 4,330,619, issued May 18, 1982; U.S. Pat. No. 4,427,777, issued Jan. 24, 1984, and 4,609,627, issued Sep. 2, 1986, all to Goldstein, all disclose methods and enzymes useful in the enzymatic conversion of red blood cells for transfusion purposes.
.alpha.-D-galactosidases EC 3.2.1.22! are a common class of exoglycosidases. Although physical properties of these enzymes differ as a group, and the physiological significance of these enzymes are not clearly established, isozymes of .alpha.-D-galactosidase are common to many plant species (1,2). Several investigators have studied .alpha.-D-galactosidase from Coffea (3). There are reports that several isozymes exist for the Coffea .alpha.-D-galactosidase enzyme (4).
Although research has been conducted with regard to the use of Coffea .alpha.-D-galactosidase for cell membrane modification studies, there has not been to date a publication throughly characterizing substrate specificity, physical characteristics, detectable protease activity, or detailed data proving homogeneity in the preparation used in those studies (5).
The Coffea canephora enzyme has special significance because of its specificity and pH optimum. The enzyme degrades the blood type B antigen creating the less immunogenic blood type O (6). Such activity can have great significance with regard to its use in the enzymatic conversion of erythrocytes from blood type B to O, thereby enlarging the compatible blood supply.
Tannins inhibit enzymes, precipitate proteins, and in the presence of oxygen, tannins generate quinones that covalently bind proteins (7). Since tannin is abundant in Coffea beans, the rapid and efficient removal of tannin resins has been determined to be desirable.
Tannin extractions from various preparations have been reported. Original methods for tannin extraction from coffee beans were described by several investigators (4,8). The tannin was removed from bean meal by sequential extractions with benzene or acetone/toluene. Acetone, toluene, and benzene are flammable; benzene is carcinogenic; and all are difficult to dispose of safely. Furthermore, several long extractions were required and it was difficult to remove all of the tannin. Preliminary experiments by Goldstein et al have shown that treatment of enzyme/tannin mixtures with certain high molecular weight polymers preserves enzymatic activity while removing tannin (7).
The present invention provides a process by which extraction of tannin from a supernatant derived from a Coffea bean homogenate results in a purified .alpha.-D-galactosidase isozyme having significantly increased activity.